Subject: Time:8:23 AM
OFFICE MEMO antisense and gene expression Date:4/11/95
Dear Friends of Chlamydomonas:
I have gotten a number of interesting responses to my query about experiences
using regulatable promoters and antisense for manipulating gene expression in
Chlamydomonas.
There have been a number of attempts at antisense by various people, some
attempts having been more extensive than others, and I think there is reason
for some optimism. I suspect that the success of this approach may depend
strongly on the particular gene whose expression is being repressed.
On the potentially negative side, Steve Gantt writes:
"I tried to use antisense RNA to block plastid chaperonin (cpn60-
alpha) gene expression. We created several antisense constructs
consisting the nitrate reductase promoter fused to different
regions of the chlamydomonas cpn60-alpha cDNA sequence. These
constructs were co-transformed along with the arg-7 gene into
arginine auxotrophs. We selected for arginine prototropy on arg
minus, ammonium-containing plates and isolated several hundred
colonies. These colonies were then replica plated onto arg minus
plates that either contained ammonium or nitrate as a nitrogen
source. I expected that cpn60 antisense co-transformants would
display altered growth characteristics (slow or no growth) on the
nitrate plates. Unfortunately, I could not convince myself that
any of the putative transformants grew slower on the nitrate
plates.
This was a one-shot experiment and shouldn't be taken to mean
that it can't work. We never even examined the transformants for
the presence of the cpn60-alpha construct or antisense
transcript. We also don't know what the phenotype of a cpn60-
alpha mutant would look like. I have assumed that a cpn60-alpha
null mutation would be lethal and cells containing low amount of
the protein would grow slowly. Similar work has been done in
yeast with the mitochondrial chaperonin."
Stefan Fabry has made rather extensive attempts to manipulate ypt gene
expression in Chlamydomonas and Volvox. His experiences are somewhat
encouraging for antisense expression, I think, and his lack of success with
expressing epitope-tagged ypt proteins may mean that such expression would be
deleterious. His description follows:
"For some time we have been trying to express antisense
as well as HA-epitope-tagged Ypt genes
(encoding small G proteins) in Chlamy (and Volvox).
(a) HA-epitope-tagged proteins: In the beginning, we used
ypt cDNA for HA-fusion, and the ars promotor from
John Davis (as you intend to do). We introduced
the construct by cotransformation together
with pArg7.8 into an Arg- strain. We verified
successful cotransformation by PCR, induced the promoter by
sulfur deprivation, and looked on Western blots
for protein synthesis with the
anti HA antibody in protein crude extracts
from arg+ cotransformants, but could not detect
any signal. We then continued
by adding an intron to the 3' region of the gene construct;
however, it also failed. We then assumed that
intron-free coding regions may in general not be
expressed in Chlamy (is that true? are there any other
experiences?), and reconstructed the whole
system with a genomic DNA. This was also not
successful.
Then we speculated that the ars promoter was not
reliable enough for our system, and used the rbc
promotor (as described in Kozminski, Diener, and Rosenbaum,
1993, Cell Motil. and the Cytoskeleton, 25, where
they expressed a HA-tagged alpha tubuline; actually
we got exactely their plasmid for our purposes).
Again, however, we did not receive any signal up to now, even
after checking now nearly 100 co-transformants.
(b) antisense:
In principle, similar experiences were made with antisense
ypt experiments. We only used the constitutive rbc promoter (even
risking that this will result in lethality of positive
cotransformants). However, we observed one time (and only
with very fresh transformants!) that a respective
Ypt protein was in fact totally absent from the
protein extract of two transformants (on the same blot,
lots of positive controls were included. However, after repeating the
experiment (after growing the strains for some
further time), the protein reappears. At moment, we
speculate that we had observed a transient antisense expression,
even though the gene construct seems to be stably integrated
within the genome.
Finally: Currently, we are having very similar
negative experiences when trying
to express (tagged and non-tagged) histone genes
in Volvox.
So to summarize, there appear some general facts,
as far as we see:
While in both algae, the cotransformation rate is quite
good, the nonselectable marker
of the cotransformation experiment, though inte-
grated in the genome, is not expressed!
The only exception I know up to now is the
successful HA-tagged-alpha-tubuline experiment
published by Rosenbaum and coworkers. My question
therefore is: does anybody have similar or other
experiences?
If my speculation is true, namely that if you do
not s e l e c t for the cotransformed gene, it will "disappear"
in a silent region of the genome with high
probability, then antisense expression of genes
(which will also be nonselectable in most cases)
may cause considerable trouble!"
The most encouraging news comes from Bernhard Scheidlmeier in repressing
arylsulfatase gene expression in Volvox. His description follows:
"To evaluate if antisense technologies are working in Volvox, I decided to
`knock out' the expression of the Volovox arylsulfatase gene. The antisense
vector consists of the Volvox ars promotor and a 2.3 kb genomic ars-gene
fragment (n-terminal region) cloned behind in inverse orientation.
So far, I have analyzed the ars enzyme activity of 7 transformants compared to
that of the non-transformed recipient strain. The results look promissing:
One transformant showed no detectable ars enzyme activity at all, the other
ones
exhibit inhibtion of ars from 60 to 95 %.
I have to repeat these mearurements some more times. So please, take these
data
only as preliminary results."
My own experience with a variety of plastocyanin mutants in Chlamydomonas is
that transformants carrying mutations that do not eliminate gene/protein
function are easy to recover. However, using genes with more extensive
deletion mutations, we have been unable to recover transformants that
accumulate RNA (or protein). This suggests a number of possibilities. One
possibility is that expression of the mutant gene product is deleterious to
the cells and that 1) transformants carrying the DNA are not recovered at all
or 2) there is strong selection for transformants that do not express or have
silenced the DNA. Further analysis should distinguish these possibilities.
I hope that we can continue this dialog and get around these troubling
technical difficulties. At the very least, a reliable, regulatable promoter
would be useful.
Karen Kindle